Note: Descriptions are shown in the official language in which they were submitted.
~2099~2
This invention relates to pharmaceutical compositions for
human and animal use in the treatmen~ and prevention of
cancerous conditions, as well as to methods for the
preparation of such compositions.
PRIOR ART
Extracts of plants of the Hypoxidaceae family have been
found to be active in in vivo scr~ening tests with
mouse P388 for the treatment of lymphocytic leukemia
('Distribution of Anticancer Activity in Higher Plants'
A. Barclay and R.E. Purdue, Cancer Treatment Reports
1976, 60 1081 - 1113). For the purposes of this
specification, lymphocytic leukemia is not regarded as a
cancerous condition, and if it is such condition is
excluded from the scope of the invention. Extracts of the
Hypoxis genus have also been used for anti-inflammatory
and prostata hypertrophy treatment (U.K. patents 1595240,
1417272, 1259503).
DEFINITIONS OF THE INVENTION
According to the invention a composition useful in
preventing or treating cancerous conditions, includes
the compound of Formula I below and in a preferred
form, includes an aqueous, alcoholic, a~ueous alcoholic,
or other extract of a plant of the Hypoxidaceae family.
~2(~99~2
It is believed that all species of the Hypoxidaceae
family are useful in this respect, but particularly
good results have been obtained using extracts from
species of the Hypoxis genus such as Hypoxis acuminata,
Hypoxis nitida, H obtusa-nitida, H. latifolia, H. rigiduli,
H. rooperi. In particular, an ethanol extract of H. rooperi
and Spiloxene schlechteri gave excellent results.
~ithout wishing to be restricted to a theory, it has
appeared to the Applicant that the anti-cancer activity
of such extraet is due to the presence of the compound:-
OH
Gluct~side-D-~-O-~ I /~
~LC = C - CE3 2 - C - C ~ -O-~-D- Glu(side
5 4 3 2 1
and its glycoside congeners.
Dosages vary from individual to indivi~ual and also on
the severity of the cancerous eondition. However it
has been found that daily dosages of from 600 mg to
240Q mg of powder obtained from extraction of plants
or 300 mg to 1200 mg of eompound (I) are effective
-- 2 --
. . ~
~2099i2
As mentioned above the extraction may be carried out
with water or an alcohol or a mixture thereof. The
following examples illustrate the methods of
extracting to the invention.
Example 1
Freshly pulped corms of Hypoxis rooperi (10 kg)
were extracted with ethanol (25Q) at room temperature
for 48 hours. Filtration, and concentration in vacuo,
followed by freeze-drying gave a brown powder (729 g~.
This crude material ~10) was separated by preparative
HPLC using a reverse phase column (PrepPAK-500/C18)
with MeOH-H20 (50:50) as solvent and gave 6.9g of an enriched
fraction. Final separation took place by separating lOg
portions of this material on a Si gel 60 column
(230-400 mesh 45mmx600mm) with butan-2-ol-C6H6-H20-MeOH
(4:3:2:1) as solvent. Typically a lOg fraction yielded
7.9g (40% from the fresh corms) of crystalline (Ij from
butan-2-ol, m.p. 147-148C, [~,-110 (~,0.45 in methanol).
(Found: C,54~18,H,6.01,Calc for C29H34014.2H20:
C,54.20,H,5.96;H205~61%);vmax(KBr?:3150-3500 (H bonded OH)
cm ~max (MeOH): 247, 257 and 287 nm; ~(CD30D?, 3.10-4.05
(14H, m, 2 protons on C-3 and 12 glucosylprotons?, 4.75
~2H,m, 2 protons on C-l?, 6.06 (H,dt,J=15.0 and
J = 5-.0 Hz, proton on C-4?, 6.53 (lH,d, J=15 Hz, proton on
C-5?, 6.65 -6.90 ~4H, m, Arom H), 7.07 (2HCd, Arom H).
.~
12~,~9~
Example 2
The following further extractions were carried out for
purposes of evalua,tion of the best methods and conditions
of extraction.
a) Gra-ted corms (10 kg) of ~Iypoxis rooperi were air dried
and the product (2.95 kg) was extracted for 1 }lo~lr with
boiling water (20 1). Spray drying of this extract
provided a partially water soluble light brown powder
(550 g), which contained 20~ of Compound (I).
] b) Gums obtained by centrifuging off the solid material
:from macerated corms (10 kg) of 1l, rooperi were diluted
with cold water (1.51, 5 C) and the centrifugate (4 1)
was spray dried to yie]d a partially water so_uble light
cream coloured powder (900 g) which contained 30% of
Compound (I).
c) Pulped corms of HyFoxis ~btusa-nitida (10 kg) were
extracted with water (15 1,25C) with occasional
stirring for 6 hours. The filtered extract (12.78 1)
was freeY.e dried to yield a partially water soluble light
brown powder (757 g) which contained 35% of Compound I.
The residual wet pulp(10.8 kg~ was extracted with
boiling abxclute ethanol (10.8 1) for 20 minutes. The
filtered extract was concentrated under vacuum to remove
most of the alcohol, diluted with water ~1 litre),
and then freeze dried to provide an almost completely
water soluble light browr, powder (361 g) which contained
43% or Compound (I).
- 4 --
120gg~2
d) I'u'l,pe(i corms of _. obt,us.l-nit,ida (lOkg) were
ext;racted with boiling wat,cr (15 1) I`or 5 minutes.
The filtrate (10.4 l) was free~,e dried -to provide a
partially water soluble light brown powder (742 g)
which contained 37% of Compourld (I).
e) Pulped corms of _. obtusa-nitida (10 kg) were
extracted with absolute ethanol (25 1, 25 C) for 66
hours with continuous stirring. The filtrate (23 1)
was concentrated under vacuum to remove most of the
alcohol, dil.uted with water (1 litre) and then freeze
dried to provide a water soluble dark brown powder ~1)
(630 g) whi,ch contained 45% of Compound (I). The
residual moist pulp (8.o5 kg) was extracted with boiling
absolute ethanol (20 1). The filtrate treated as
lS described above was freeze dried to provide an almost
completely water soluble dark brown powder (ii) (99 g)
which contained 42% of Compound (I).
f) Thinly sliced corms of H. obtusz.-obtusa ((:L~kg)
were freeze dried to provide dried material (3.086 kg).
This dried material was milled and a portion (300 g)
was extracted with boiling 50% aqueous ethanol (5 1)
for 5 minutes. The filtrate was treated as described
above and freeze dried to provide a water soluble light
cream powder (l'L6 g) which contained 35% of Compound (I).
g) Pulped corms of young H. obtusa-nitida were
extracted with boiling water ~5 1) for 5 minutes. The
fiLtrate on freeze drying provided a partially water
soluble light brown powder (54 g) which contained
~d 32% of Compound ~I).
- 5 --
9~12
h) An extraction on young l-l. obtusa--nitida corms as
described under (g) above, but pro'Longing the boiLing
process to 20 minutes, resultecl in a re:Latively ins()lu~le
dark hrown ~owder (82.3g) whi,ch contained 27% of
Compound (I).
i) Fr~sh _ypoxis acuminat;a corms are pulped directly
into ethanol at room temperature (1.5 kg corms per 2
ethanol). After stirring the pulp for 1 hour it is
fiLtered. The fi:ltrate is evaporated under vacuum
(30 Kpa ! at elevated temperatures (70-85C). The
obtained residue is freeze dried to provide a yellow-
brown product in 7 - 10% yie:Ld from the fresh corms.
The content of' I on this extract varies between
~2 - 57% l'hese yields depend on seasonal v.~riations.
j) Fresh llypoxis acuminata corms are sliced directly
irto liquid nitrogen. The fro~en slices are free~e-
dried and milled to a fine powder. This powder is
then extracted with aqueous alcohol (1 part water to
2 parts of ethanol vr methanol) using 6e of the solution
per 1 kg of plant powder. After 30 minutes stirring
the mixture is filtered. Evaporation of the solution
frcm the filtrate as described in example 2i provides
a yellowish-brown product in 9 - 12% from the original
fresh corms. The content of I of this extract vari,es
between 49-56%. Yields depend on seasoral variati.ons.
~2~99~2
k. Pulped corms of Spiloxene schlechteri on treatment
with aqueous ethanol provided an extract which contained
a substantial amount of Compound (I).
The above evalutations were obtained by comparative thin
layer chromatography and HPLC analysis.
"It will be seen that exceptionally good yields are
obtained in the above examples when the solvent is aqueous
methanol or ethanol and the extraction takes place at low
temperature for a short period of time. This result is
contrasted with the results obtained heretofore - for
example in our Canadian Patent 9~9,388 wherein the examples
3(a), 3(b) and 3(c) produced no measurable or only very
small yields of sterolin and the particular method of
extraction was abandoned. In fact, it was not realized
at that time that these methods resulted in a large yield
of another compound, namely the compound of the present
invention."
Example 3
In order to demonstrate the activities of the extracts of
the invention, cytotoxicity tests were carried out on
M(52)B cell culture systems in vitro.
-
---` 1209912
Mouse Sarkoma cells M(52)B were cultured as monolayers
in M.E.~I. with 106 foetal calf serum. After confluency was
achieved (usually within 3-4 days depending on the number
of cells added per bottle) the cells were trypsiniz~d.
Once loose the cell suspension was treated with M.E.M.
containing 10% foetal calf serum in order to inactivate the
trypsin.
An arbitrary volume of the suspension was then transferred
to fresh nutrient solution rendering a final volume of
- 10 5 cm3
- 7a -
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~20~1912
The test material was added at different concentrations
to eitller the freshly prepared cell suspensi-,n or to the
24 hour developed cell culture in which the monolayer had a
confluency o~ at lease 40 to 50%
All procedures were carried out under sterile conditions
in a laminar flow chamber.
RESULTS
'l`he addition of adcquate amounts of the test material
:resulted in cell death within 24 hours. In other words
the transferred cells failed to attach themselves on the
bottom surface of the tissue culture flask, while the
untreated control cells l`ormed a collLluent m~n(Jlaye
within 48-72 hours.
When less than adequate amounts of the test material
were added some regeneration of the cells became
apparent in the culture flasks over the first 24 hours.
However, in these cases it took more than 10 days to
achieve a 40-60% confluency, indicating that the
larger proportion of the transferred cells had been
comp]etely destroyed.
-- 8
. .~,
~20
rl .1 1> ] e :
Percentage confluency due to H. obtusa-nitida
(extracts ex example 2e(i)
Observation period in days
Dose confluency in %
Agent mg/ml 1 3 6 10 14 Days
Control nil 60 90 at 4s _
_ _
Extract 0.4 few -5 -10 +4~ 30-80
f`rom grow
example ing
2e(i) cells
:.- o.8 (?) n/g n/g n/g +
1.6 n/g n/g n/g n/g n/g
2.4 n/g n/g n/g n/g n/g
n/g No observed growth, with only isolated cells
attached to the bottom surface of tissue culture flask.
The extracts of the invention may be administered as such
but preferably in the form of their spray-dried or
freeze dried powders, in which case they may be provided
in the form of tab:Lets, capsules, dragees, creams, powders,
ointments and any other usual form of medicament. The
recognised additives may also be present to improve
tabletting, capsuling and the like. Dosage units may
]o contain from approximately 100 mg to approximately
50 mg of extract per unit dose, and the rate of
administation is 1 to 3 units, 3 times per day.
~20s~æ
Example 4a
Acute tox;city eva]uation of' t,he Hypoxis ext,ract.
-
Toxicity tests were carried out with solutions
prepared from extracts as described in example 2e(i)
using groups of mice (equal numbers of males and
females).
The weight of the animals ranged between 22 and 30g.
The follow-up observation period extended over 7 days
following the administation of a single dose per animal.
The prepared solutions were filtered through Millex
filter membranes (.45mm) before they were administered
intravenollsly in order to remove any particles which may
complicate the interpretation of the results, and to
obtain a sterile so:Lution.
For the oral or intraperitoneal route the solutions were
tS administered as prepared, i.e. without prior filtration.
The results are presented in Table 2.
-- 10 --
Table 2
-
Cumulative percentage mortality due to H.obtusa-nitida
extract (example 2e(i)
Cumulative ~0 mo.tality
Route Days
Dose of
mg/kg admin 1 2 3 5 7
500 P.0 0 0 0 0 0
1000 P . O O O O O O
2000 P.0 0 0 0 0 0
_
500 I.P 0 0 0 0 0
1000 I.P 10 20 20 20 20
2000 I.P 60 75 80 80 80
_ .
500 I.V 0 0 0 0 0
1000 I.V 20 25 25 25 25
2000 I.V 80100 _
Example 4b
Acute toxicity evalua-tion of ~I)
Solutions o~ (1) in dist,illed water were prepared
with the required concentration. Administered
volumes were kept constant at the rate of 10 cm3per kg.
In each case equal numbers of males and females were
treated with a single dcse. All animals were observed
over 7 days for signs of toxic and/or pharmaco-dynamic
effects.
-- 1 1 --
~æo~
~necropsy was performed on all surviving animals.
Results are presented in the Tab]e.
..
Table, 3
Acute toxicity eva:luation of (I~ on oral,
intraperitoneal, and intravenous admi~istration.
_ ---
~pecies Administration I)ose Mortality Remarks
Mode mg/k (Range %)
.. _ _ _ I
Mollse Oral lOOO 0/20 No macroscopic
2000 0/20 lesions were found
4000 0/20 in any animal
_
~ouse Intra- 250 O/lO Following admin-
peritoneal 500 0/20 istration all anima]
lOOO 5/30(10-30) exhibited signs of
2000 7/30(80-lOO discomfort which
increased in degrees
. _ ~ ~ with increasing
Rabbits Intravenous 50 0/2 No vis.ible effects
lOO 0/2 were observed and
no macropscopic
iesions were found
in these animals.
No thrombophlebitis
was found at the
_ _ inJection site.
Conclusions:
It appears that ( T ) exhibits a low degree of direct toxic
effects. No macroscopic lesions were observed on necropsy
of the surviving animals.
~209912
,
Example 4c
Foetotoxic - Teratogenic effect of (I)
Two groups of six fully impregnated mice (Swiss Webster
offspring) were treated orally with aqueous solutions of
(I) once per day (10 ml/kg) starting day 1 through to day
8 of the gestation period. On day 20 of the pregnancies the
mice were lightly anaethetised with diethyl ether and the
uterus exposed by caesarian section. The total number of
implantation sites were counted. All foeti were removed
and examined for (a) number alive and
(b) macroscopic gross anatomical abnormal-
ities of limbs and head.
The results were compared to a control group and are
presented in Table 4.
.
Table 4
Effects of orally administered (I) on pregnant mouse foeti
G~OUP CONTROL COMPOUND I
H20 only20 mg/kg 100 mg/kg
No of animals 6 _ _ ~ 6
No of implantation sites 56 58 58
No of-foeti 52 52 49
No of live foeti 51 50 45
No of malformations1 ~?) 3 (2?)
,~ - 13 -
Example 4~d)
"In vitro" cytostatic,~cytotoxic effects with
Hypoxis rooperi extracts (examp:le 2(e)(i~)and Compound I
Four different cel]. culture types were used:
i. Mouse sarcoma -- Ms 52 B
ii. Mouse mela~oma - Me~ Bl - 6
iii. Hella 229.
iv. ~uman foetal foreskin fibroblasts - IIFSF`
(non-malignant)
Procedure Trypsinized cells were harvested into MEM
and suitably d-i]uted to supply about 2x105 cells per cm3.
~ culture flask containing 4,5 cm3Of culture medium
was seeded with 0.5 cm3 of the prepared cell suspension.
Thi.s cell culture was then incuba-ted at 37 C f'or 24 hours
hefore the test substances dissolved in 0.5 cm3 of the
culture medium were added. A1,1 cultures were grown
u~til an apparent 100% was achieved in the contro'lflasks..
In all cases presented in TABL~ 5 cell growth was
retarded considerably and growth regression was observed
for MS 52 B. Mel Bl - 6 and 11eha 229 cell cultures.
- 14 -
~æ
TABLE, S
-
Incubation temp 37 C Confluency
_
Cell culture type MS 52 B Mel Bl - 6 lleha 229 HFSF
test material
amounts added in Ext. I Ext. I Ext. I Ext. I
ng 80 4080 40 80 $0 100 40
_ _.
Hours: 24 5 4 40 40 50 5 40 40
48 4020 40 30 50 60 fiO ~0
72 2020 20 4~ 40 50 75 50
96 2020 5 40 20 3 60 40
l20 - - - 30 - - - -
. .
In all cases controls had reaehed 100%
confluency by 96 hou/rs.
Ext. = Hy~oxis rooperi extract as prepared
in exampLe 2e~i)
I -- pure Compound I as described in
example 1.
~ 5
~zoss~æ
Despite the small number of animals tested it seems
unlikely that at the doses administered (I) exhibits
teratogenic effects. Th~re were no indications of
abnormally reduced grow~h rate of the foeti in any of
the treated groups. No adverse effects were detected
in the treated mother animals.
Example 5
Hypoxis extracts prepared as described in Example 2
were used to treat individual patients with various
types of cancer, with the following surprising results.
a) A 64 year old male patient who had suffered fro-m
skin cancer (face and arms) had developed an inoperable
growth near his anus and had been operated on for growths
on his cheek and lips. After treatment had commenced
with an extract prepared as described in Example 2a
(200 mg doses three times per day3 his condition
improved in that all cancerous growth started to
regress within the first three weeks to finally disappear
completely. The patient was treated six years ago and
he is still alive and well.
2Q b) The medical case history of a man, now aged 79, reads
as fol-lows:-
At age 7~ the patient had a laparotomy and colostomy.
~indi-ngs: a huge mass in the sigmoid colon, rectosigmoid
~unction and rectum. Due to extensive peritoneal
infiltration of the tumour and the fact that
obstruction was present it was decided to biopsy
- 16 -
iZ09~12
several of the excrescences and to perform proximal
co],ostomy. The pathology leport recorded the presence
of lymphosarcoma and this was :Later confirmed by an
~-ray investigation. Surgery was recommended to be
following by post-operative radiation treatment.
The surgeon on performi~ a~ exploratory laparotomy
found a huge mass within the sigmoid colon and upper
rectum. It had infiltrated onto the po,sterior
abdominal and lateral pelvic walls making a hard
10 unresectible mass. After this operation the patien-t
was not expected to live longer than two months. At
this stage treatment commenced with a Hypoxis rooperi
extract as described in Examples 2a a~ ,b with a daily
d~se of 3 times 200 mg. Four months later the patient was
15 still alive and underwent surgery to free the colostomy
-from the abdominal wall and carry out a large bowel
resection; no additional abnormalit,ies were found in
the rest of the peritoneal cavity. A pathology report
on the excised 'intussuscepted' large bowel section recorded
20 that t,he intussusGeption was oedematus and indurated
(hardened) while the histology report records that sections
of this bowel specimen showed congestion and superficial
ulceration of the mucosa, but no evidence of lymphoma
or malignancy. Since then the patient has continued
25 to use llypoxis extracts intermittently and he has
regained his weight. He is now 79 years old and
is active and healthy. There are no signs of a
'recurrence of the cancer.
- 17 -
~a.s~z
c) An inoperable lung cancer was diagnosed in a middle
aged female seven years ago. She commenced with a
Hypoxis ~ extract prepared as described in
example 2a wit,h a dose of 600 mg per day. She
received no chemotherapy ~r radiation treatment.
Wit,hin a month the patient reported a subjective
improvement and wi-thin six months the pati~nt had
recovered completely. Thi~: patient is still alive,
she takes the llypoxis extract regularly and reports at
least -twicc a yeal.
.) A bed-ridden male patient urlderwent radiation
treatment for an inoperable lung cancer and he was
not expected to survive for lt~nger than two months.
Trcatment with llypoxis roopeli extract prepared as
descrihed in Example Za and 2b using 600 mg per day
resulted in an improvement enabling the patient to
leave his bed and walk around. He was still alive
six months later when due to lack of communication,
treatmcnt with the extract was discon-tinued. Within
two weeks the patient had to return to his bed.
Trea-tment was resumed and within a week the patient was
hell enough to walk around again. Four months later this
patient again discontinued treatment and he died shortly
thereafter.
e) A 33 year old female patient after a radical
mastectomy followed by radiation and chemotherapy
developed metastatic cancer in the spine resulting
- 18 -
, , .
~9~2
in the destruction ot` one vertebra. ~urther
chemotherapy was lefused by the patient who commenced
treatment with a Hypoxis obtusa-llitida extract
-
prepared as described in Example 2e(i) at a dose
5 of 3 x 300 mg per day.
A month later a bone scan was performed, no sign
of the cancer could be found and the patient was
operated on within a week to rebuild the ver~tebra
with a hip-bone transpLant. l~To sign of a residual
L0cancer cou]d be found during th,s operation.
f) Two females both sufferin~ from severe skin
cancer were treated with a Hypoxis rooperi extract
prepared as described in Example 2a at a dose ot`
3 x 200 mg per day. Their condition cleared up
15within 60 days.
- 1 9 -
~' .
~0,9g~
Example 6
Two female patients with histo-pathological cor.firmed
carcinomas were treated with 600 mg Hyooxis extract
4 times per day. ~oth patiel:ts were considered incurable
when the l~y~oxis extract treatments commenced and they
then received no other cytostatic drugs or radiation
treatment other than medication to relieve their
general state of debilitation and pain. The l-lypoxis
extract contained 52% (I) and was prepared from
H. acuminata as described in example 2e i.
a) A ?7 year old patient had an infiltrating growth
of a solid mammary carcinoma with pleural metastases
and with distinc:t effusion. After 64 days treatment
with the l~ypoxis extract regression of the effusion was
obvious together with a reduction of the
metastases; there had been no need for drainage.
The overall condition of thc patient had improved
and she reported to be completely free from pain.
b) A 54 year old patient had a serious papillary
ovarian adeno-carcinoma accompanied by peritoneal and
mesen-terial infiltrations as well as ascites. After
2 2 months treatment with the l~ypoxis extract the
general condition of the patient had improved. The aescites
had regressed without the aid of drainage or di~retica.
- 20 -
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